Fasciculation in the early mouse trigeminal nerve is not ordered in relation to the emerging pattern of whisker follicles

Detailed reconstructions of lengths of the embryonic mouse maxillary nerve were made from serial light and electron microscope sections to determine whether there is any correspondence between the arrangement of fasciculi in the developing nerve and the emerging pattern of whisker follicles on the snout. There was neither correspondence nor obvious pattern in the arrangement of fasciculi at either E11, when trigeminal nerve fibers first contact the presumptive whisker pad, or at E12, when the pattern of developing whisker follicles becomes apparent. Fasciculi merged and branched to form an intricate plexus. Furthermore, the ratio of the number of nerve fibers in one fasciculus to the number in another prior to their merger differed significantly from the ratio of fiber numbers in the two fasciculi after their separation, which indicates that nerve fibers are freely exchanged between fasciculi. Our findings suggest that the somatotopic representation of the whisker follicle pattern in the brainstem does not develop by nerve fiber growth augmenting an initially ordered pattern of fasciculi.     

Influence of nerve growth factor on developing dorso-medial and ventro-lateral neurons of chick and mouse trigeminal ganglia

Trigeminal ganglia have been removed from five, six, seven and eight day chick embryos and explants of the dorso-medial (DM) and ventro-lateral (VL) parts of the maxillomandibular lobe were grown in tissue culture. Quantitative methods were used to assess the influence of nerve growth factor (NGF) on fiber outgrowth from these explants. At all ages outgrowth from DM explants was significantly greater than from VL explants, the difference being most pronounced between the extreme DM and VL poles of the maxillomandibular lobe. These observations are interpreted as indicating the existence of two distinct populations of neurons in terms of their response to NGF rather than the consequence of the asynchronous differentiation and maturation of the VL and DM neurons. A similar study of 10, 11 and 12 day embryonic mouse trigeminal ganglia revealed no significant difference in neurite outgrowth between DM and VL regions grown in the presence or absence of NGF.     

Sympathetic ingrowth to the trigeminal ganglion following intracerebroventricular infusion of nerve growth factor

The objective of the present study was to examine the remodeling of uninjured sympathetic axons in the adult rat trigeminal ganglion following a 2-week in vivo intracerebroventricular infusion of NGF. The accumulation of infused NGF in the trigeminal was assessed using ELISA and sympathetic fibers were localized immunohistochemically with an antibody to tyrosine hydroxylase (TH). In addition, high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) allowed for biochemical measurements of the catecholamines norepinephrine (NE) and dopamine (DA). Increased NGF protein in the trigeminal ganglion was paralleled by a significant increase in sympathetic fibers and pericellular plexuses (i.e. baskets) in the cell body regions. Some ganglia showed elevated NE following NGF infusion, yet the 88% increase in mean NE did not reach significance. Following bilateral removal of the sympathetic superior cervical ganglia (SCG), a significant reduction was observed in overall NE levels and in TH-immunoreactive (-ir) fibers in the cell body regions and peripheral branches, suggesting the SCG as the origin of the sympathetic ingrowth. However, mean DA levels as well as TH-ir fibers within the trigeminal central branch were unaffected by NGF infusion or removal of the SCG and likely resulted from intrinsic dopaminergic cell bodies. In conclusion, our data provide evidence that the increased availability of NGF in the young adult rat trigeminal ganglion observed following in vivo NGF infusion enhanced sympathetic associations with the sensory neurons in the trigeminal, supporting a role for NGF in the regulation of sympathosensory interactions.     

The effects of nerve growth factor (NGF) and antiserum to NGF on the development of embryonic sympathetic neurons in vivo

The role of nerve growth factor (NGF) in the development of embryonic sympathetic neurons was examined in vivo. Individual mouse embryos received transuterine injections of NGF or antiserum to NGF (anti-NGF), and the effects on the superior cervical ganglion (SCG) were studied. Treatment with NGF at any gestational stage, from the time of ganglion aggregation to birth, increased ganglion tyrosine hydroxylase (T-OH) activity. Both the number of catecholaminergic neurons and T-OH activity per neutron were increased. Choline acetyltransferase (ChAc) activity was increased by NGF at early gestational stages, but not at later stages. These observations suggest that perikarya containing ChAc are responsive to NGF, whereas preganglionic nerve terminals are not. Treatment with anti-NGF rapidly and permanently decreased ganglion T-OH activity. The effects of anti-NGF were more pronounced at later gestational stages, suggesting that ganglia become increasingly dependent on NGF during development. Alteration of maternal levels of NGF had no effect on development of the embryonic SCG, suggesting that local embryonic concentrations of NGF are responsible for modulating sympathetic ontogeny.     

The effects of exogenous nerve growth factor on foetal rat adrenal cells in culture

Summary Foetal rat adrenal cells, identified as chromaffin cells, maintained in culture in the presence of exogenous NGF, express a neuronal phenotype. Once this neuronal phenotype has been established bundles of outgrowing neurites commonly form links with adjacent reaggregates, and also develop synaptic contacts with the cell bodies of chromaffin cells in the adjacent reaggregates. Subsequent withdrawal of exogenous NGF from the culture medium did not result in the chromaffin cells losing their neuronal phenotype if this had been established before NGF was withdrawn. Tyrosine hydroxylase immunocytochemistry revealed that all neurites developed in culture were positive for the enzyme including those in cultures only transiently exposed to exogenous NGF. These results suggest that exogenous NGF may be essential only until the chromaffin cells develop neurites which make contact with appropriate target cells and that once these links, however immature, have been established, neurites are retained for many weeks in the absence of exogenous NGF.     

Effect of nerve growth factor on the elongation of neurites from axotomized rat embryonic septal-basal forebrain neurons: an in vitro analysis

The administration of nerve growth factor (NGF) into the brain of a fornix-fimbria lesioned rat can rescue many cholinergic, septal-basal forebrain (SBF) neurons from imminent cell death. Unfortunately, it is unclear if NGF can stimulate regenerative growth from axotomized, SBF neurons. In the present study, we used an in vitro model system to determine if NGF could affect neurite outgrowth from nonaxotomized and/or axotomized, embryonic SBF neurons. Axotomized neurons were obtained by severing the neuritic fields surrounding embryonic day (E) 15 SBF explants maintained in primary culture. Acetylcholinesterase (AChE) histochemistry was used to assess the effects of NGF on cholinergic neurites. We report that 1) neurite outgrowth on type I collagen from E15 SBF neurons in primary culture (nonaxotomized neurons) was not affected by NGF. 2) NGF enhanced the outgrowth (regeneration) of axotomized, SBF neurons on a collagen substratum; however, neurons had to be treated with NGF both before and after axotomy to stimulate regeneration effectively. Application of NGF either before or after axotomy did not enhance regenerative neurite outgrowth. 3) SBF neurons had to be axotomized for NGF to facilitate neurite outgrowth. This is supported by the observation that SBF explants, initially maintained in NGF-supplemented medium in suspension culture, did not demonstrate enhanced neurite outgrowth in the presence of NGF when plated onto a substratum. 4) The regenerative growth of AChE-negative, as well as AChE-positive, neurites was facilitated by NGF treatment. In addition to data concerning neurite outgrowth, we also found that the NGF receptor, as recognized by the antibody 192-IgG, expands its distribution as time in culture progresses; i.e., staining, originally confined to cell bodies and proximal processes within the explant, later included neurites that emanated from the explant. Thus, our results demonstrate that NGF can stimulate regenerative growth from axotomized, but not nonaxotomized, embryonic SBF neurons. We hypothesize that, given the appropriate substratum for axon elongation in vivo, NGF can stimulate the regeneration of SBF neurons in the injured adult brain.     

Sensitivity of NGF-responsive dorsal root ganglion neurons to semaphorin D is maintained in both neonatal and adult mice

Using a coculture assay of DRG neurons and aggregates of cells transfected with individual semaphorins, we have investigated the ability of semaphorins A, D, and E to inhibit axonal growth from DRG neurons. We show that axons of these neurons that grow in response to NGF remain responsive to semaphorin D in neonatal and in adult mice, although sensitivity may decline in the latter. Consistent with these findings, expression of the semaphorin receptor, neuropilin-1, is maintained in the DRGs of adult mice.     

Reduced neuronotrophic activity of fibroblasts from individuals with dysautonomia in cultures of newborn mouse sensory ganglion cells

The neuronotrophic activity of human skin fibroblast cell lines from two normal individuals and 3 individuals with Familial Dysautonomia (FD) was compared in terms of their ability to support neuron survival and neurite regeneration in cultures of newborn mouse sensory neurons. Neuronotrophic activity was assayed by culturing dissociated sensory neurons from newborn mice: (1) on fibroblast 'cell beds' consisting of monolayer cultures of living fibroblasts, (2) with medium that had been conditioned by monolayer fibroblast cultures and (3) with extracts of the cultured fibroblasts. Neurite regeneration was compared by determining the percentage of neurons that had regenerated neurites after 24 or 48 h in culture. Neuron survival was determined by counts at 48 h, 7 days and 14 days after culture initiation. Neurite regeneration and neuron survival was found to be significantly less on FD cell beds or with FD-conditioned medium than in replicate cultures with normal fibroblasts or their conditioned medium. The reduced neuronotrophic activity of FD fibroblasts or conditioned medium was still observed in the presence of antibody sufficient to block the neuronotrophic effects of purified nerve growth factor (NGF). Thus, fibroblasts from individuals with FD exhibit reduced neuronotrophic activity for newborn mouse sensory neurons that appears to be due to factor(s) other than NGF.     

Bovine normal peripheral nerve and nerve sheath tumour in explant culture

Summary When bovine normal nerve and nerve sheath tumours were cultured using explant techniques, the same morphological cell types were seen: thin bipolar cells with bulging nuclei; round refractile cells; large quadrangular cells; variable polar, wide-processed cells. However, differences in the pattern of cell growth and the proportions of morphological cell types occurred between normal and tumour nerve.The bovine nerve sheath tumours cultured showed a range of histological areas which did not appear to affect the pattern of cell growth in culture or the cell types present. Antoni type A tissue was prominent in many explant blocks and probably gave rise to most cell growth.On comparison with human schwannomas maintained in explant culture, bovine nerve sheath tumours appeared to have a similar range of morphological cell types. Although suggestions for the origin of human schwannomas have been made on the basis of cell morphology, it was felt that morphology alone did not allow speculation on the origin of cells present in bovine nerve sheath tumour explant cell growth.This work was supported by grants from the Australian Meat Research Committee, the George Aitken Pastoral Trust and the Cancer Research Committee of the University of Sydney     

Loss of ganglion cells in fetal retina transplanted to rat cortex

Fetal retina survived and differentiated when placed into the cortex of newborn host rats. Two months after transplantation, anterograde tract tracing techniques failed to reveal any projections from these transplants into the host brain. Soma-size analysis of the cells in the ganglion cell layer of the transplants suggested that only displaced amacrine cells remained and that the ganglion cells had degenerated. The basis for these results is considered relative to trophic maintenance of ganglion cells, retinal axon guidance, and specificity of connections.     

Spontaneous voltage and current fluctuations in tissue cultured mouse dorsal root ganglion cells

Fetal mouse dorsal root ganglion (DRG) neurons were maintained in primary dissociated cell culture for periods of 7 days to 3 months. Intracellular recordings from these cells revealed the presence of spontaneous subthreshold potentials in 101/177 neurons studied. When measured at the resting membrane potential, these spontaneous voltage events took two forms: (a) high frequency potential fluctuations several millivolts in peak-to-peak amplitude and (b) small, discrete hyperpolarizations. Neurons exhibiting either type of event were designated as 'active' DRG cells. No spontaneous potentials were seen in DRG cells hyperpolarized to membrane voltages more negative than -64 +/- 11.5 mV (n = 5 cells). Under voltage-clamp conditions, the subthreshold potentials of active DRG cells were replaced by fluctuations in outward current. The power spectral density, S(f) of these current fluctuations was approximated by an equation of the form S(f) = (S(o)/[1 + (f/fc) alpha] where 2 less than or equal to a less than or equal to 3 and the half-power frequency fc = 11.3 +/- 3.1 Hz at 23 degrees C (n = 17 cells). The spontaneous voltage fluctuations of active DRG cells were abolished in Ca2+-free saline, and of the divalent metal cations Sr2+, Mg2+, Ba2+, Co2+ and Mn2+, only Sr2+ could substitute for Ca2+ in the maintenance of this activity. Tetraethylammonium ions (1-10 mM) reversibly blocked the spontaneous potentials, while caffeine (10 mM) increased the frequency of these events. The spontaneous voltage fluctuations were not dependent on the presence of spinal cord neurons in the culture plate, and they were also observed in cultured DRG cells derived from adult mice.     

Increased serotonin in the developing superior colliculus does not alter the number or distribution of retinotectal ganglion cells

Administration of a single subcutaneous dose of 5,7-dihydroxytryptamine (5,7-DHT) to newborn hamsters results in a significant increase in the density of serotoninergic (5-HT) fibers in the superficial layers of the superior colliculus (SC) and marked abnormalities in both the crossed and uncrossed retinotectal projections when these animals reach adulthood (R. Rhoades, C. Bennett-Clarke, R. Lane, M. Leslie, and R. Mooney, 1993, J. Comp. Neurol. 334:397–409). The present study was undertaken to determine whether changes in the retinotectal projection of 5,7-DHT-treated animals were associated with alterations in the number or distribution of retinal ganglion cells in these animals. Nissl staining of retinae from normal adult and 5,7-DHT-treated hamsters revealed no differences between them in the number or average diameter of cells in the retinal ganglion cell layer. Retrograde labeling with horseradish peroxidase (HRP) demonstrated no effect of 5,7-DHT treatment on the number or distribution of ipsilaterally or contralaterally projecting ganglion cells. Neonatal 5,7-DHT administration also had no effect on the distribution of soma diameters for HRP-labeled retinal ganglion cells. Electron microscopic analysis demonstrated no significant difference between the number of optic nerve fibers in the normal and 5,7-DHT-treated hamsters. The results are consistent with the conclusion that the effect of 5,7-DHT on the retinotectal projection may primarily be a function of this toxin, or the increase in 5-HT it induces, on the terminal arbors of retinotectal axons rather than on their parent cells. © 1996 Wiley-Liss, Inc.     

Amelioration of delayed neuronal death in the hippocampus by nerve growth factor

Selective neuronal death in the CA1 sector of the hippocampus [delayed neuronal death (DND)] develops several days after transient global cerebral ischemia in rodents. Because NGF plays a potential role in neuronal survival, it was decided to study its effect in DND. We report here that intraventricular injection of NGF either before or after 5 min forebrain ischemia in the Mongolian gerbil significantly reduced the occurrence of DND. The tissue content of NGF in the hippocampus was decreased 2 d after ischemia and recovered to the preischemic level by 1 week. By the Golgi staining technique, changes first began in the dendrites of affected neurons as early as 3 hr. Such changes could be ameliorated by NGF treatment. Although previous knowledge of NGF is limited to the survival of cholinergic neurons in the CNS, it is assumed that other mechanisms must be operating in the hippocampus, for example, postsynaptic modification at dendrites or aberrant expression of NGF receptors possibly at the initial excitation period by glutamate. Furthermore, because previous work has shown that inhibition of protein synthesis reduces the occurrence of DND, a program leading to cell death might also be operating via de novo synthesis of certain protein(s), collectively termed "killer protein," because of a lack of NGF.     

Principal neurons as local circuit neurons in the rat superior cervical ganglion: The synaptology of the neuronal processes revealed by intracellular injection of biocytin

To analyze the local circuitry of the sympathetic ganglion, the synaptic relations of the neuronal processes of the principal neurons in the rat superior cervical ganglion were investigated by correlated light and electron microscopy combined with intracellular injection of biocytin. Intracellular iontophoresis of biocytin followed by avidin-biotinylated horseradish peroxidase cytochemistry allowed complete visualization of the neuronal processes of the principal neurons. The stained principal neurons have a single process (axon), which leaves the ganglion, and several intraganglionic processes (dendrites), some of which show specific terminal arborizations. Some terminals of the dendritic collaterals formed pericellular plexuses or intercellular glomerular plexuses. Electron microscopically, the dendrites and their collaterals contain numerous small vesicles. Synaptic membrane specializations were observed between the stained dendritic collaterals and unlabeled neurites. These may be both preganglionic axon terminals and processes of principal neurons. The likely direction of neurotransmission often could not be determined because of the bidirectional synaptic structures. Our findings show that the dendritic collaterals of principal neurons appear to make both post- and presynaptic contacts with both the principal neurons and the preganglionic axons. It is suggested that the principal neurons might participate in local circuits involving not only preganglionic axons but also neighboring principal neurons. © 1993 Wiley-Liss, Inc.     

Ultrastructure of the ganglion cells of the terminal nerve in the dwarf gourami (Colisa lalia)

In our previous light microscopic studies (Oka et al., Brain Res. 367: 341-345, '86; Oka and Ichikawa, J. Comp. Neurol. 300: 511-522, '90), we reported that there are at least two types of terminal nerve (TN) cells based on cell size and immunoreactivity: type I cells had large cell bodies, while type II cells had smaller cell bodies. Type I TN cells were immunoreactive to gonadotropin-releasing hormone (GnRH) and may be the major source of GnRH-immunoreactive fibers that are widely distributed throughout the brain. Type II TN cells, on the other hand, were not immunoreactive to GnRH. In the present paper, we examined the cytology and synaptology of these two types of TN cells with electron microscopy. Type I TN cell bodies were found to have morphological characteristics similar to those of other peptide-synthesizing neurons and are likely to be actively synthesizing GnRH. The frequent occurrence of coated vesicles close to the plasma membrane of the cell body was suggestive of membrane retrieval following exocytosis of the vesicular contents from the cell surface. Neighboring TN cells were either in direct juxtaposition with one another or made specialized "glomeruloid" cell-to-cell contacts; these specializations may be relevant for nonsynaptic intercellular communications among the TN cells. Within these glomeruloid complexes, the somatic processes of TN cells received inputs from two types of synaptic terminals: one containing only spherical synaptic vesicles and another containing a small number of dense-cored vesicles in addition to the spherical synaptic vesicles. Axosomatic synapses were rare on type I TN cell bodies. In contrast, type II TN cell bodies had morphological characteristics similar to those of neurons in other brain regions. These receive axosomatic inputs from synaptic terminals containing only spherical synaptic vesicles and those with a small number of dense-cored vesicles in addition to the spherical synaptic vesicles. Thus, each type of TN cell has unique fine structural characteristics which may correlate to their different functional roles.     

On the neuronal origin of the afferents to the ciliary ganglion in cat

The histological constitution of the oculomotor nuclear complex and the surrounding structures of cat was re-examined, and it was found that the so-called Edinger-Westphal (EW) nucleus and the anteromedian nuclei consisted of oval cells of small sizes and polygonal cells of medium sizes. The latter cells were scattered in the peripheral portions of the EW and the anteromedian nuclei, and were frequently intermingled with cells of the same type scattered in the central gray and the medial portion of the ventral tegmental area (VTA). Horseradish peroxidase (HRP) was injected into the ciliary ganglion, in order to locate the cells of origin of afferents to this ganglion. The cells labeled by an injection into the ganglion were these polygonal cells of medium sizes scattered in the injected side of midbrain. They weremainly distributed in the VTA, the central gray, and the caudal half of the anteromedian nucleus. The peripheral portion of the EW and the anteromedian nuclei also contained some labeled cells, but these were considered to be cells which had migrated from the central gray and the VTA. Most of the cells in the EW and the anteromedian nuclei were labeled by the injection into the spinal cord. Hence, it was concludedthat the main sources of the afferents to the ciliary ganglion were polygonal cells of medium sizes scattered in the VTA and the central gray, and some of these intermingled with the cells of peripheral boundaries of the EW and the anteromedian nuclei. By contrast, the greater part of the EW and the anteromedian nuclei had a connection with the spinal cord, but not with ciliary ganglion.     

Ultrastructure of retinal ganglion cell death after axotomy in chick embryos

Axotomy often leads to neuronal death, which occurs after a particularly short delay in immature animals. Tectal lesions were made in embryonic day (E) 12 chick embryos, thereby axotomizing the retinal ganglion cells of the contralateral eye, which then died within 3 days. We here describe the ultrastructural changes in the axotomized ganglion cells. The main changes were nuclear invagination and type 3B (cytoplasmic type) cell death characterized by dilation of the perinuclear space, endoplasmic reticulum, and Golgi apparatus. However, nuclear invagination was never seen in type 3B dying cells. All the axotomy-induced retinal ganglion cell death appears to have been of type 3B; apoptosis was not induced by axotomy, as was confirmed by additional light microscopic experiments showing that it did not increase the frequency of apoptotic markers revealed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (the TUNEL method) labeling and immunoreactivity for activated caspase-3. However, the latter methods did show small numbers of apoptotic cells dying naturally even in control retinas. After the death of the axotomized ganglion cells, they were phagocytosed mainly in Müller processes. The present findings open up the chick tectal lesion model as a system for analyzing type 3B neuronal death in vivo.     

Sciatic nerve transection produces death of dorsal root ganglion cells and reversible loss of substance P in spinal cord

Sciatic nerve section has been shown to reduce substance P (SP) in the dorsal horn of the spinal cord, but the mechanism which underlies the reduction is not understood. Whether SP levels subsequently recover as they do after dorsal rhizotomy has also been unknown. To test the hypothesis that transganglionic degeneration of primary afferents contributes to the reduction of SP, we have studied the changes in SP which result from section of the cat sciatic nerve and determined the extent of dorsal root ganglion (DRG) cell death. Sciatic nerve section resulted in DRG cell death, but the amount was variable and not seen in all animals. Reduction in dorsal horn and DRG SP was seen in all animals, and in the spinal cord it was followed by recovery. These sequelae resemble the changes which follow dorsal rhizotomy. After sciatic nerve section, the reduction in dorsal horn SP is small than after rhizotomy, the recovery more complete, and both the reduction and the recovery proceed more slowly. Evidence is presented that similar mechanisms may contribute to depletion of intraspinal SP after sciatic nerve section and after dorsal rhizotomy. The mechanisms contributing to recovery of spinal cord SP after sciatic nerve section may resemble known mechanisms of recovery that occur when the lesion is central.     

A quantitative electron microscopic analysis of the infraorbital nerve in the newborn rat

The infraorbital nerve (n = 3) was examined in newborn rats using electron microscopic techniques. Counts of the entire nerve revealed an average of 42,051 (S.D. = 2083) unmyelinated and 168 (S.D. = 47) myelinated fibers. The unmyelinated axons averaged 0.46 μm (S.D. = 0.16) in diameter while the myelinated fibers averaged 1.71 μm (S.D. = 0.17).